Plant Growth Promotion and Salt Stress Alivation by IAA Producing Endophytic Fungi

Abstract

In the present study 43 endophytic fungi were isolated from different parts of two xeric plants, Solanum xanthocarpum and Euphorbia milli. Colonization frequency for different parts of two plants was checked. S. xanthocarpum and E. milli showed 73.97% and 66.7% endophytes isolation frequency respectively. Similarly, the endophytic infection rate of S. xanthocarpum and E. milli was 67.57% and 67% respectively. These isolates were also checked for their growth promoting activity on maize plant treated with yucasin. The salt resistant potential of all the selected isolates were also investigated by adding 100mM NaCl, K2SO4, KCl to Czapek broth. The fungal biomass (g) and IAA (ug/mL) were determined and compared with control having just Czapek broth inoculated with the fungal isolates. The fungal isolates, FH1, FH2, FH3, FH4, FH5, FH6, FH7, FH8, FH9, FH10, FH11, FH12, FH13, FH14, and FH15 were observed to produce halozones around the colony on Pikovskaya’ s media. Most of the fungal isolates were phosphate solubilizers and were able to produce ammonia. The ability of endophytic fungi to produce phytohormones and secondary metabolites like IAA, phenols and flavonoids offers a valuable opportunity to select plant growth promoting endophytic fungi. The isolates were screened for IAA production and 15 strains were able to produce tryptophan dependent IAA. The endophytes that screened positive for tryptophan dependent IAA production were identified on the basis of 18s rDNA gene sequence homology. The isolated fungal endophytes belonged to eight different genera i.e. Alternaria, Fusarium, Yarrowia, Candida, Ceratocystis, Corynespora smithii, Penicillium, Aspergillus and Meyerozyma. The tryptophan dependent IAA biosynthesis was further confirmed by the presence of tryptophan hdependent IAA biosynthetic pathway (Indole-3-Acetamide) genes (IaaM and IaaH) in the isolated fungi i.e. Aspergillus sp., Candida membranifaciens, Yarrowia sp. (reported for the first time) and Fusarium oxysporum. The preliminary detection of IAA was done through spectrophotometer, but the quantification of IAA was confirmed through GC/MS SIM and HPLC. Total phenols and flavonoids were also quantified through spectrophotometer. An incubation time and tryptophan concentration dependent increase in IAA production was observed. Most efficient tryptophan dependent IAA producers were Yarrowia lipolytica FH7, Yarrowia lipolytica FH11, Aspergillus flavus FH12, Yarrowia lipolytica FH13, Fusarium oxysporum FH14 & Candida membranifaciens FH15. They produced IAA in the range 26-50 µg/mL. Different carbon sources (sucrose and glucose) and nitrogen sources (peptone and yeast xvii extract) were used to optimize IAA production by the endophytes. Sucrose and peptone were the most suitable sources of carbon and nitrogen enabling the endophyte to produce significantly high amount of IAA. It was noted for the first time that phenols and flavonoids production by endophytes was tryptophan concentrations dependent. The optimum concentration of tryptophan was 500ug/mL, where further increase was detrimental for a number of endophytes with few exceptions. Media containing sucrose and peptone were optimum for the production of phenols and flavonoids by most of the fungal strains including FH1, FH3, FH4, FH5, FH6, FH7, FH8, FH11, FH12 and FH13. As our aim was to use the isolated strains for salt stress alleviation in plants, it was imperative to check their DPPH radicals scavenging activity. To our knowledge it was the first ever report on the presence of DPPH radical scavenging activity in fungi including of Alternaria, Fusarium, Yarrowia, Candida, Ceratocystis, Penicillium and Meyerozyma. In most of the fungal strains DPPH scavenging activity was high when sucrose and peptone were used as carbon and nitrogen sources respectively. The response of fungi in terms of DPPH scavenging activity was variable to the increasing concentration of tryptophan in culture media. It was observed that salinity (100mM NaCl) drastically reduced all the growth parameters of maize. Among the selected isolates, P. citrinum (FH1), A. alternata (FH2), Y.lipolytica (FH3), Y. lipolytica (FH4), P. oxalicum (FH5), Y. lipolytica (FH11), Y. lipolytica (FH13), F. oxysporum (FH14) and M. caribbica (FH15) enhanced the growth of maize seedlings under salinity. All the growth attributes including chlorophyll, carotenoids, plant fresh and dry biomass, shoot and root length, relative water content, proline content, peroxidase, catalase and IAA content were increased significantly after endophytes application under all treatments. Mitigation of salt stress in maize seedlings by the endophytes was evident from normal or near normal electrolytes leakage in salt stressed maize. An increase in the activities of SOD, CAT, and proline due to salt stress was also observed. Mitigation of salinity stress in maize seedlings after endophytes application was observed in terms of further increase in anti-ROS. Presence of all these valuable properties makes the fungal isolates good plant growth promoters to be used as biofertilizers to improve growth and yield of crops.